Conductive graphene matrix-encapsulated cells

ABSTRACT

Various embodiments disclosed relate to conductive graphene matrix-encapsulated cells. A matrix-encapsulated cell includes an encapsulating polymer matrix including a biopolymer and graphene. The matrix-encapsulated cell also includes one or more of the cells encapsulated within the encapsulating polymer, wherein the graphene directly contacts at least some of the cells. The matrix encapsulating the one or more cells is electrically conductive.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.16/716,250, filed Dec. 16, 2019, which claims the benefit of priority toU.S. Provisional Patent Application Ser. No. 62/808,018 filed Feb. 20,2019, the disclosures of which are incorporated herein in their entiretyby reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under N000141612246,N000141712620, and W911NF1710584 awarded by the Department of Defense.The U.S. Government has certain rights in this invention.

BACKGROUND

Hydrogel microfibers have many applications in tissue engineering andregenerative medicine, where they are favored for their physical andchemical properties, as well as their reproducible and cell-safefabrication methods. A variety of biocompatible polymers are utilizedfor this method of microfiber creation; among them, alginate is favoredwithin biomedical applications for its good biocompatibility,biodegradability and low toxicity, as well as its capacity forpolymerization within mild conditions. These factors have garneredinterest for alginate in the realms of cell encapsulation, whichrequires cells to be present during the gelation of the microfibers,thereby eliminating the possibility of cell loss but requiring cell-safegelation conditions. Hydrogel scaffolding creates a physiologicallyrelevant platform for studying cell behavior. Existing research hascreated hydrogels with enhanced conductivity for the purpose ofdelivering electrical stimulation to study or control cell response,viability, and regeneration potentials; however, conductivebiocompatible hydrogels remain underutilized as 3D electro-sensing cellculture scaffoldings.

While there are a range of ways to enhance the conductivity ofmaterials, only some are suitable for biomedical applications. Since itsdiscovery in 2004, graphene has drawn much attention in the fields ofnanoscience, and has become known as a functional material in biomedicalapplications due to its biocompatibility, high conductivity, andmechanical properties, which are preferable to other compounds such asreduced graphene oxide. However, challenges arise when non-toxic aqueoussolutions of graphene are required, as is the case when both cells andgraphene are encapsulated within a hydrogel.

Chemically, graphite exfoliation to form graphene can be assisted by theinclusion of suitable surfactants, which reduce interfacial tensions toaid in suspension. Unfortunately, due to graphene's hydrophobic nature,water alone is not capable of forming a stable, homogeneous solution, aspristine graphene nanosheets are subject to van der Waals forces andshow unwanted aggregation. Surfactants typically used to aid in this aretypically highly toxic.

Common mechanical techniques for graphene dispersion involve usingsonication, but this method requires additional materials andelectrochemical procedures to maintain a stable aqueous graphenesolution, which affects the biocompatibility of the resulting graphenesolution. Graphene oxide may be reduced either thermally or chemically;however, the desired characteristics of the synthesized graphene may notbe easily maintained, and requires extensive use of cytotoxic chemicalsand procedures.

SUMMARY OF THE INVENTION

Various embodiments provide a matrix-encapsulated cell. Thematrix-encapsulated cell includes an encapsulating polymer matrixincluding a biopolymer and graphene. The matrix-encapsulated cell alsoincludes one or more of the cells encapsulated within the encapsulatingpolymer. The graphene directly contacts at least some of the cells. Thematrix encapsulating the one or more cells is electrically conductive.

Various embodiments provide a fiber including an encapsulating polymermatrix including a biopolymer and graphene. The fiber also includes oneor more cells encapsulated within the encapsulating polymer. Thegraphene directly contacts at least some of the cells. The matrixencapsulating the one or more cells is electrically conductive.

Various embodiments provide a method of making the matrix-encapsulatedcell. The method includes polymerizing a pre-polymer solution, thepre-polymer solution including the one or more cells, the graphene, anda precursor for the biopolymer.

Various embodiments provide a method of using the matrix-encapsulatedcell. The method includes detecting electrical signals from or sendingelectrical signals to the one or more cells through the encapsulatingpolymer matrix.

Various embodiments provide various advantages over other encapsulatedcells, at least some of which are unexpected. For example, in variousembodiments, the encapsulated polymer matrix is substantially free ofgraphite, graphene oxide, or reduced graphene oxide, which can havelower electrical conductivity than graphene. In various embodiments,despite the use of non-conductive hydrogels in the encapsulating matrix,the increased conductivity can allow for the elucidation of electricalcell-to-cell communication mechanisms within neuronal cell cultures. Invarious embodiments, spatially restricting the location of cells duringexperiments can enable the long-term study of cell-to-cell communicationwithout risk of cells flaking away. In various embodiments, thematrix-encapsulated cells are a physiologically relevant platform forreal-time 3D conductivity measurements, thereby allowing for rapiddetection of cells' responses to chemical or mechanical inputs.

In various embodiments, the encapsulated cells can survive theencapsulation without harm and can be maintained in an encapsulatedstate while alive for extended periods. In various embodiments, thecells can be recovered from the encapsulating matrix in a living andhealthy state.

In various embodiments, a microfluidic technique can be used to form thematrix-encapsulated cell. The microfluidic technique can avoid harm tothe cells during the encapsulation. The ionic cross-linking microfluidicfiber fabrication technique utilizes an ionic exchange between fluids inlaminar flow within microfluidic chambers to create gentlepolymerization conditions that can yield tunable microfibers whichencapsulate live cells. In various embodiments, the microfluidictechnique can be versatile, allowing precise control over the diameterand cross-sectional shape of the microfiber through the ability to varymicrochannel device size and geometry, as well as the flow rate ratio(FRR) between the core and sheath fluids. Different flow rate ratios canbe used to impact the characteristics of the fibers, affecting theirsize and shape, as well as their mechanical and electrical properties.The microfluidic fabrication technique can provide gentle polymerizationconditions and tunable control over cells' spatio-temporal locations.

In various embodiments, the matrix-encapsulated cell can be in the formof a conductive fiber that encapsulates the one or more cells.Conductive materials in biomedical fields include fibers, fibrous matsformed using electrospinning, and conductive hydrogels. Currentconductive hydrogels in biomedical fields take the form of membranes,gels, or films. Conductive microfibers, particularly those whichencapsulate the cells they are trying to stimulate or study, haveuntapped potential in terms of long-term experiments where cell locationmust be controlled. Microfibers can have the ability to mimic spatiallyorganized 3D environments with controllable cell density for extendedperiods of time. Cutting-edge breakthroughs in the fabrication ofbiocompatible and stable aqueous graphene solutions enable theencapsulation of both graphene and cells within the alginate hydrogel,thereby creating a highly powerful real-time sensing platform.

BRIEF DESCRIPTION OF THE FIGURES

The drawings illustrate generally, by way of example, but not by way oflimitation, various embodiments of the present invention.

FIG. 1 illustrates a diagram of a microfluidic device used to formfibers, in accordance with various embodiments.

FIGS. 2(A)-(B) illustrate scanning electron microscopy images of fibersformed using a microfluidic technique, in accordance with variousembodiments.

FIG. 3 illustrates a plot showing fiber diameter versus flow rate ratiofor fibers formed using a microfluidic technique, in accordance withvarious embodiments.

FIGS. 4(A)-(D) illustrate rat PC12 cells seeded onto 6 well platesobserved at (A-B) 24 and (C-D) 48 hours after introduction of 200 μL of5% Graphene solution, in accordance with various embodiments.

FIGS. 5(A)-(I) illustrate rat PC12 cells in a non-polymerized control ofalginate, gelatin, graphene (A, D, G), encapsulated in alginate fibers(B, E, H), and cells which migrated from the hydrogels and attached onthe surface of the well plate (C, F, I), in accordance with variousembodiments. Day 0 (A-C), day 3 (D-F), and day 7 (G-I).

FIG. 6 illustrates cells recovered from encapsulation within 40%alginate, 10% gelatin and 1 mg/mL graphene gels after one week, with ascale bar is 100 microns, in accordance with various embodiments.

FIGS. 7(A)-(C) illustrate fibers made with alginate, graphene, and cellswithin a Microfluidic device with a flow rate ratio of 600:40μL/min:μL/min (sheath:core), in accordance with various embodiments.Cells were stained with GPA fluorescent proteins (A), imaged inbrightfield (B), and the images were combined (C). Scale bars represent100 microns.

FIG. 8(A) shows tensile stress versus tensile strain for pure alginate,alginate and graphene, and for alginate/cells/graphene, in accordancewith various embodiments.

FIG. 8(B) shows the Young's modulus for alginate, alginate and cells,alginate and graphene, and for alginate/cells/graphene, in accordancewith various embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of thedisclosed subject matter. While the disclosed subject matter will bedescribed in conjunction with the enumerated claims, it will beunderstood that the exemplified subject matter is not intended to limitthe claims to the disclosed subject matter.

Throughout this document, values expressed in a range format should beinterpreted in a flexible manner to include not only the numericalvalues explicitly recited as the limits of the range, but also toinclude all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited. For example, a range of “about 0.1% to about 5%” or “about 0.1%to 5%” should be interpreted to include not just about 0.1% to about 5%,but also the individual values (e.g., 1%, 2%, 3%, and 4%) and thesub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within theindicated range. The statement “about X to Y” has the same meaning as“about X to about Y,” unless indicated otherwise. Likewise, thestatement “about X, Y, or about Z” has the same meaning as “about X,about Y, or about Z,” unless indicated otherwise.

In this document, the terms “a,” “an,” or “the” are used to include oneor more than one unless the context clearly dictates otherwise. The term“or” is used to refer to a nonexclusive “or” unless otherwise indicated.The statement “at least one of A and B” or “at least one of A or B” hasthe same meaning as “A, B, or A and B.” In addition, it is to beunderstood that the phraseology or terminology employed herein, and nototherwise defined, is for the purpose of description only and not oflimitation. Any use of section headings is intended to aid reading ofthe document and is not to be interpreted as limiting; information thatis relevant to a section heading may occur within or outside of thatparticular section.

In the methods described herein, the acts can be carried out in anyorder without departing from the principles of the invention, exceptwhen a temporal or operational sequence is explicitly recited.Furthermore, specified acts can be carried out concurrently unlessexplicit claim language recites that they be carried out separately. Forexample, a claimed act of doing X and a claimed act of doing Y can beconducted simultaneously within a single operation, and the resultingprocess will fall within the literal scope of the claimed process.

The term “about” as used herein can allow for a degree of variability ina value or range, for example, within 10%, within 5%, or within 1% of astated value or of a stated limit of a range, and includes the exactstated value or range.

The term “substantially” as used herein refers to a majority of, ormostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or100%. The term “substantially free of” as used herein can mean havingnone or having a trivial amount of, such that the amount of materialpresent does not affect the material properties of the compositionincluding the material, such that about 0 wt % or mg/mL to about 5 wt %or mg/mL of the composition is the material, or about 0 wt % or mg/mL toabout 1 wt % or mg/mL, or about 5 wt % or mg/mL or less, or less than,equal to, or greater than about 4.5 wt % or mg/mL, 4, 3.5, 3, 2.5, 2,1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about0.001 wt % or mg/mL or less, or about 0 wt % or mg/mL.

As used herein, the term “polymer” refers to a molecule having at leastone repeating unit and can include copolymers.

Matrix-Encapsulated Cell

Various embodiments provide a matrix-encapsulated cell. Thematrix-encapsulated cell includes an encapsulating polymer matrix thatincludes a biopolymer and graphene. The matrix-encapsulated cell canalso include one or more of the cells encapsulated within theencapsulating polymer. The graphene can directly contact at least someof the cells. The matrix encapsulating the one or more cells can beelectrically conductive.

The encapsulating polymer matrix can have any suitable form, such as amembrane, gel, film, fiber, or a combination thereof. In someembodiments, the encapsulating polymer matrix has the form of a fiber.The fiber can have any suitable dimensions, such as length and diameter.As the fibers can be generated via a continuous process, the length isnot particularly limited. For example, the fiber can have a length ofabout 1 micron to about 100 meters, or about 1 cm to about 100 cm, orabout 1 cm to about 10 cm. The fiber (e.g., dried fiber) can have adiameter of about 1 micron to about 100 microns, about 2 microns toabout 50 microns, or about 10 microns to about 25 microns.

The biopolymer can be any suitable biopolymer, such as gelatin,chitosan, polycaprolactone, a polysaccharide, alginate, or a combinationthereof. The biopolymer can include calcium alginate (e.g., alginatecrosslinked via calcium ions). The biopolymer can form any suitableproportion of the encapsulating polymer matrix, such as about 0.5 mg/mLto about 10 mg/mL, about 1 mg/mL to about 5 mg/mL, or about 1.5 mg/mL toabout 3 mg/mL of the encapsulating polymer matrix. Graphene is a singlelayer of carbon, while graphite is multiple layers of graphene.

In various embodiments, the graphene is homogenously distributed in theencapsulating polymer matrix. The graphene can provide the electricalconductivity of the encapsulating polymer matrix. The graphene cancontact at least some of the cells, and the graphene particles can alsocontact adjacent graphene particles in the matrix, such that anelectrical connection is formed throughout the encapsulating matrix thatconnects to the contacted one or more cells. In some embodiments, theencapsulating polymer matrix can be substantially free of graphite,graphene oxide (e.g., similar to graphene but having more oxidizedgroups), reduced oxidized graphene (e.g., a material which is similar tographene but has more oxidized groups and therefore a different chemicalstructure), or a combination thereof. The graphene can form any suitableproportion of the combination of the encapsulating polymer matrix andthe one or more cells, such as about 5 mg/mL to about 35 mg/mL, about 10mg/mL to about 25 mg/mL, or about 14 mg/mL to about 18 mg/mL.

The one or more cells can be living cells that can be encapsulated forany suitable time period while still in a living state. In variousembodiments, the one or more cells can be recovered from theencapsulating polymer matrix in a living and healthy state. The one ormore cells can include any suitable type or variety of cells, such asneural cells, astrocyte cells, or stem cells. The one or more cells caninclude rat PC12 cells, mouse astrocyte cells (MACs), adult hippocampalprogenitor stem cells (AHPCs), or mesenchymal stem cells (MSCs). The oneor more cells can include mammalian cells. The one or more cells caninclude rat PC12 cells. The one or more cells can form any suitableproportion of the combination of the encapsulating polymer matrix andthe one or more cells, such as about 1×10² cells/mL to about 1×10¹⁰cells/mL, about 1×10⁴ cells/mL to about 1×10⁸ cells/mL, about 1×10⁵cells/mL to about 1×10⁷ cells/mL, or about 1×10⁶ cells/mL to about 5×10⁶cells/mL.

In some embodiments, the encapsulating matrix further includes gelatin.In some embodiments, the encapsulating matrix is substantially free ofgelatin. The gelatin can form any suitable proportion of theencapsulating matrix, such as about 0 mg/mL, or such as about 0.5 mg/mLto about 10 mg/mL, about 1 mg/mL to about 5 mg/mL, or about 1.5 mg/mL toabout 3 mg/mL.

The encapsulating matrix can further include a surfactant. Thesurfactant can be any suitable surfactant, such as Tween (e.g., apolysorbate-type nonionic surfactant formed by the ethoxylation ofsorbitan before the addition of lauric acid, such as Polysorbate 20(polyoxyethylene (20) sorbitan monolaurate), Polysorbate 40(polyoxyethylene (20) sorbitan monopalmitate), Polysorbate 60(polyoxyethylene (20) sorbitan monostearate), or Polysorbate 80(polyoxyethylene (20) sorbitan monooleate)), polyethylene glycol (PEG),or bovine serum albumen (BSA). The surfactant can include bovine serumalbumen (BSA).

In some embodiments, the encapsulating matrix can further includesolvents in addition to water, such as polyethylene glycol (PEG).

Method of Making the Matrix-Encapsulated Cell

Various embodiments provide a method of making the matrix-encapsulatedcells. The method can include polymerizing a pre-polymer solution. Thepre-polymer solution can include one or more cells, graphene, and aprecursor for the biopolymer. The method can be a microfluidictechnique, or can be another technique. The pre-polymer solution can bean aqueous solution.

The graphene can be non-agglomerated and form any suitable proportion ofthe pre-polymer solution. For example, the graphene can be about 1 mg/mLto about 30 mg/mL, about 10 mg/mL to about 30 mg/mL, about 15 mg/mL toabout 25 mg/mL, or about 18 mg/mL to about 22 mg/mL of the pre-polymersolution.

The precursor for the biopolymer can form any suitable proportion of thepre-polymer solution. For example, the precursor for the biopolymer(e.g., alginate) can be about 1 mtg/mL to about 15 mg/mL, about 2 mg/mLto about 8 mg/mL, or about 3 mg/mL to about 5 mg/mL of the pre-polymersolution.

The pre-polymer solution can further include a surfactant to maintainthe graphene in a non-agglomerated state during the polymerization. Thesurfactant can be any suitable surfactant. The surfactant can be about 1mg/mL to about 20 mg/mL, 5 mg/mL to about 15 mg/mL, or about 8 mg/mL toabout 12 mg/mL of the pre-polymer solution. In some embodiments, thesurfactant can include Bovine Serum Albumen (BSA). Bovine serum albumencan be used to aid with the dispersion of graphene. BSA is awater-soluble protein which is able to make non-covalent bonds with bothpositively and negatively charged particles, a feature which aids in thestability, encapsulation efficiency, and release rates in the field ofdrug delivery. BSA bonds with graphene non-covalently, thereby enablingthe creation of a highly stable, non-aggregating aqueous graphenesolution that may be stored in ambient conditions over extended periods.

In some embodiments, the pre-polymer solution further includes gelatin.In other embodiments, the pre-polymer solution is substantially free ofgelatin. The gelatin can form any suitable proportion of the pre-polymersolution, such as about 0.5 mg/mL to about 10 mg/mL of the pre-polymersolution.

In some embodiments, the pre-polymer solution can include solvents inaddition to water, such as polyethylene glycol (PEG). The PEG can formany suitable proportion of the pre-polymer solution, such as about 0.1mg/mL to about 50 mg/mL, about 1 mg/mL to about 5 mg/mL, or about 2mg/mL to about 3 mg/mL. PEG can reduce agglomeration of the graphene,and has both low toxicity and efficiency as a solvent for carbon-carbonbonds. In various embodiments, a microfluidic core-sheath technique forforming the fibers can include PEG in the core solution while the sheathsolution is substantially free of PEG.

The polymerizing of the pre-polymer solution can include exposing thepre-polymer solution to a crosslinking solution. The crosslinkingsolution can be any suitable solution that induces crosslinking of theprecursor for the biopolymer. For example, the crosslinking solution caninclude an aqueous Ca²⁺ solution, and the precursor for the biopolymercan be alginate, with the biopolymer formed being calcium alginate.During the crosslinking, the Ca²⁺ molecule can be diffused into thepre-polymer solution, thereby creating a calcium alginate encapsulatingpolymer matrix.

The polymerization can include injecting the pre-polymer solution intothe crosslinking solution. The polymerization can include exposing thepre-polymer solution to the crosslinking solution in a microfluidicdevice. Performing the method using a microfluidic device can produce ahydrogel microfiber with highly tunable conductivity and mechanicalproperties.

The method can include forming a non-agglomerated aqueous solution ofgraphene. In order to fabricate a non-toxic aqueous solution ofgraphene, both chemical and mechanical manipulation of graphite can beused, such as exfoliation and fragmentation of graphite throughsonication and magnetic stirring in BSA. Exfoliation may take the formof sonication, ultrasonics, ball milling, or a combination thereof.

Method of Using the Matrix-Encapsulated Cell

Various embodiments provide a method of using the matrix-encapsulatedcell. The method can include detecting electrical signals from orsending electrical signals to (e.g., detecting, measuring, or acombination thereof) the one or more cells through the encapsulatingpolymer matrix. Detecting the electrical signals from the one or morecells can include detecting responses of the one or more cells tochemical or mechanical stimulus applied to the one or more cells.

EXAMPLES

Various embodiments of the present invention can be better understood byreference to the following Examples which are offered by way ofillustration. The present invention is not limited to the Examples givenherein.

Example 1. Preparation of Graphene Solution

A solution of 2% BSA, 5% Graphite, and water was prepared using ballmill. Similarly, a 0.1 mg/mL solution of graphene was fabricated throughpure sonication. This graphene solution was used in the Examples herein.

In an alternative technique, a solution of 1,000 mg graphite and 500 mgBSA and water is prepared using a ball mill.

Example 2. Preparation of Alginate/Graphene/PEG Core Solution

3% Alginate and 2.5% PEG (w/v) was dissolved into the Graphene solutionby magnetic stirring overnight at 800 rpm. The solution was sterilizedvia long-term exposure to UV radiation. Similarly, a solution of 40%Alginate and 10% Gelatin was used for extrusion encapsulations.

Example 3. Biocompatibility Test

To test the biocompatibility of the graphene solution, 200 μL wereintroduced into cells seeded onto a 6 well plate in 1 mL of media. Thesecells were observed for 48 hours.

Example 4. Fabrication of Microfluidic Devices

Microfluidic devices were created by curing PDMS on a photolithographicmold bearing the design. These PDMS halves were joined using plasmacleaning, and inlets were attached via glue.

Example 5. Fabrication of Alginate/Graphene/PEG Microfibers

Sheath solution was prepared with a concentration of 0.5% CaCl₂ and 5%PEG (w/v) in DI water. The core solution (i.e., the pre-polymersolution) was introduced into the center channel of the microfluidicdevice, while the sheath is pumped through the two outer channels. Asyringe pump was used to maintain constant fluid velocity of 600:40μL/min:μL/min (sheath:core). Resulting fibers were introduced into a 5%CaCl₂ water bath before collection. FIG. 1 illustrates a diagram of themicrofluidic device.

The sheath solution traveled through the microfluidic device with thecore solution, shaping it and helping to make sure it did not clog. Anionic transfer (e.g., calcium ions) from the sheath solution polymerizedthe core solution. As compared to extrusion, the microfluidic techniqueprovided greater control over the shape and size of the fiber. Themicrofluidic technique may also help to align particles and bonds in away that does not occur during an extrusion technique.

In an alternative technique, the flow rate ratio is 255:55 μL/min:μL/min(sheath:core). This flow rate ratio was used with a core solution of 3%alginate, 1.75% PEG, and 2.2% graphene. FIGS. 2(A)-(B) show scanningelectron microscopy images of the generated fibers. FIG. 3 shows a plotshowing fiber diameter versus flow rate ratio, showing both long andshort diameters, since the cross-sectional area of the fiber was notperfectly round

Example 6. Fabrication of Alginate/Graphene/Gelatin Extruded Microfibers

Rat PC12 cells were introduced into the alginate/gelatin/graphenesolution and were injected into a 10% CaCl₂ bath through a needle withan inner diameter of 0.013 mm. The resulting fibers were gathered andplaced into 12 well plates for observation.

Example 7. Recovery of Encapsulated Cells

Cells were recovered by removing polymerized cell-laden alginate gelsand placing them into 1 mL of 0.1 M PBS solution. Gels were gentlyaspirated, after which they were allowed to rest at 37° C. for 10minutes. After one final aspiration, the resulting solution wascentrifuged and the resulting cell suspension was plated.

Example 8. Mechanical Properties of Fibers

Fibers were created and mounted onto paper frames for transport. Theywere analyzed using an Instron Universal Testing Machine (Model 5569,Instron Engineering Corp., Canton, Mass.) with a 10 N load cell and anextension rate of 1 mm/min.

Example 9. Microscopy

Images were collected with an Axio Observer Z1 Inverted Microscope fromZeiss. Initial processing such as contrast and brightness were completedwithin the AxioVision Special Edition 64-bit software. Furtherprocessing, such as removal of debris outside of the well plate andcompiling of fluorescent cell images, was completed within AdobePhotoshop CC 2018.

Example 10. SEM Images

(SEM) analysis was performed using a JCM-6000 NeoScope Benchtop SEM withan accelerating voltage of 15 kV.

Example 11. Analysis of Results Example 11.1. Biocompatibility ofGraphene

Graphene introduced to cells seeded within six well plates did not causeany apparent sign of cellular distress. Cells were still adherent after48 hours of graphene interaction, as seen in FIGS. 4(A)-(D), showing ratPC12 cells seeded onto 6 well plates observed at (A-B) 24 and (C-D) 48hours after introduction of 200 μL of 5% Graphene solution.

Example 11.2. Encapsulation of Rat PC12 cells with Graphene

Rat PC12 cells were successfully encapsulated within graphene-ladenalginate/gelatin fibers via the extrusion method, as seen in FIGS.5(A)-(I), showing day 0 (A-C), day 3 (D-F), and day 7 (G-I); (A, D, G)non-polymerized control of alginate, gelatin, graphene and rat PC12cells; (B, E, H) cells encapsulated in alginate fibers extruded in a 10%CaCl₂ bath from a needle with an inner diameter of 0.013 mm; and (C, F,I) cells which migrated from the hydrogels and attached on the surfaceof the well plate. Cells were observed migrating from within the fiberbody to attach to the bottom of the well plates. This shows that cellssurvived the encapsulation process and maintained their ability toproliferate in standard conditions. Likewise, the unpolymerizedalginate/graphene control (A, D, G) further show the biocompatibility ofgraphene over a seven-day process.

Cells encapsulated in this way were successfully recovered after oneweek, as seen in FIG. 6 . Cells survived within the polymer, and uponrecovery showed the ability to adhere to the surface of well plates andproliferate. Cells maintained standard morphologies.

Example 11.3. Microfluidic encapsulation of Rat PC12 cells inGraphene/Alginate Solutions

Cells and graphene were successfully encapsulated within Alginate fibersusing a microfluidic device. FIGS. 7(A)-(C) illustrate fibers made withalginate, graphene, and cells within a microfluidic device with a flowrate ratio of 600:40 μL/min:μL/min (sheath:core). Cells were stainedwith GPA fluorescent proteins (A), imaged in brightfield (B), and theimages were combined (C). Scale bars represent 100 microns.

Example 11.4. Characterization of Fibers

Fibers were characterized and their elastic moduli were calculated.Preliminary data shows minor differences in the mechanical properties offibers made with pure alginate fibers, alginate and graphene fibers, andalginate/graphene fibers with encapsulated cells. FIG. 8(A) showstensile stress versus tensile strain for pure alginate, alginate andgraphene, and for alginate/cells/graphene. FIG. 8(B) shows the Young'smodulus for alginate, alginate and cells, alginate and graphene, and foralginate/cells/graphene. Preliminary results of adding graphene andcells to microfluidically created 1.5% alginate fibers in a 5% CaCl₂bath and a flow rate ratio of 225:10 μL/min (sheath:core) showed thatthese factors do not affect mechanical properties.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theembodiments of the present invention. Thus, it should be understood thatalthough the present invention has been specifically disclosed byspecific embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those of ordinaryskill in the art, and that such modifications and variations areconsidered to be within the scope of embodiments of the presentinvention.

Exemplary Embodiments

The following exemplary embodiments are provided, the numbering of whichis not to be construed as designating levels of importance:

Embodiment 1 provides a matrix-encapsulated cell comprising:

-   -   an encapsulating polymer matrix comprising a biopolymer and        graphene; and    -   one or more of the cells encapsulated within the encapsulating        polymer, wherein the graphene directly contacts at least some of        the cells;    -   wherein the matrix encapsulating the one or more cells is        electrically conductive.

Embodiment 2 provides the matrix-encapsulated cell of Embodiment 1,wherein the encapsulating polymer matrix is a fiber, a fibrous mat, amembrane, or a film.

Embodiment 3 provides the matrix-encapsulated cell of any one ofEmbodiments 1-2, wherein the encapsulating polymer matrix is a fiber.

Embodiment 4 provides the matrix-encapsulated cell of any one ofEmbodiments 1-3, wherein the biopolymer comprises gelatin, chitosan,polycaprolactone, alginate, a polysaccharide, or a combination thereof.

Embodiment 5 provides the matrix-encapsulated cell of any one ofEmbodiments 1-4, wherein the biopolymer comprises a polysaccharide.

Embodiment 6 provides the matrix-encapsulated cell of any one ofEmbodiments 1-5, wherein the biopolymer comprises calcium alginate.

Embodiment 7 provides the matrix-encapsulated cell of any one ofEmbodiments 1-6, wherein the biopolymer is about 1 mg/mL to about 5mg/mL of the encapsulating polymer matrix.

Embodiment 8 provides the matrix-encapsulated cell of any one ofEmbodiments 1-7, wherein the encapsulating polymer matrix issubstantially free of graphene oxide.

Embodiment 9 provides the matrix-encapsulated cell of any one ofEmbodiments 1-8, wherein the encapsulating polymer matrix issubstantially free of reduced oxidized graphene.

Embodiment 10 provides the matrix-encapsulated cell of any one ofEmbodiments 1-9, wherein the graphene is homogeneously distributed inthe encapsulating polymer matrix.

Embodiment 11 provides the matrix-encapsulated cell of any one ofEmbodiments 1-10, wherein the graphene is about 10 mg/mL to about 25mg/mL of the combination of the encapsulating polymer matrix and the oneor more cells.

Embodiment 12 provides the matrix-encapsulated cell of any one ofEmbodiments 1-11, wherein the graphene provides the electricalconductivity of the fiber.

Embodiment 13 provides the matrix-encapsulated cell of any one ofEmbodiments 1-12, wherein the one or more cells are one or more livingcells.

Embodiment 14 provides the matrix-encapsulated cell of any one ofEmbodiments 1-13, wherein the one or more cells comprise rat PC12 cells,mouse astrocyte cells (MACs), adult hippocampal progenitor stem cells(AHPCs), or mesenchymal stem cells (MSCs).

Embodiment 15 provides the matrix-encapsulated cell of any one ofEmbodiments 1-14, wherein the one or more cells comprise mammaliancells.

Embodiment 16 provides the matrix-encapsulated cell of any one ofEmbodiments 1-15, wherein the one or more cells are about 1×10⁴ cells/mLto about 1×10⁸ cells/mL of the combination of the encapsulating polymermatrix and the one or more cells.

Embodiment 17 provides the matrix-encapsulated cell of any one ofEmbodiments 1-16, wherein the one or more cells are about 1×10⁶ cells/mLto about 5×10⁶ cells/mL of the combination of the encapsulating polymermatrix and the one or more cells.

Embodiment 18 provides the matrix-encapsulated cell of any one ofEmbodiments 1-17, wherein the one or more cells comprise rat PC12 cells.

Embodiment 19 provides the matrix-encapsulated cell of any one ofEmbodiments 1-18, wherein the encapsulating matrix further comprisesgelatin.

Embodiment 20 provides the matrix-encapsulated cell of any one ofEmbodiments 1-19, wherein the encapsulating matrix further comprises asurfactant.

Embodiment 21 provides the matrix-encapsulated cell of Embodiment 20,wherein the surfactant comprises polyoxyethylene (20) sorbitanmonolaurate, polyoxyethylene (20) sorbitan monopalmitate,polyoxyethylene (20) sorbitan monostearate, or polyoxyethylene (20)sorbitan monooleate), polyethylene glycol (PEG), bovine serum albumen(BSA), or a combination thereof.

Embodiment 22 provides the matrix-encapsulated cell of any one ofEmbodiments 20-21, wherein the surfactant comprises bovine serum albumen(BSA).

Embodiment 23 provides the matrix-encapsulated cell of any one ofEmbodiments 1-22, wherein the encapsulating matrix further comprisesPEG.

Embodiment 24 provides the matrix-encapsulated cell of any one ofEmbodiments 3-23, wherein the fiber has a diameter of about 1 micron toabout 100 microns.

Embodiment 25 provides the matrix-encapsulated cell of any one ofEmbodiments 3-24, wherein the fiber has a diameter of about 10 micronsto about 25 microns.

Embodiment 26 provides a fiber comprising:

-   -   an encapsulating polymer matrix comprising a biopolymer and        graphene; and    -   one or more cells encapsulated within the encapsulating polymer,        wherein the graphene directly contacts at least some of the        cells;    -   wherein the matrix encapsulating the one or more cells is        electrically conductive.

Embodiment 27 provides a method of making the matrix-encapsulated cellof any one of Embodiments 1-25, the method comprising:

-   -   polymerizing a pre-polymer solution, the pre-polymer solution        comprising        -   the one or more cells,        -   the graphene, and        -   a precursor for the biopolymer.

Embodiment 28 provides the method of Embodiment 27, wherein the grapheneis about 10 mg/mL to about 30 mg/mL of the pre-polymer solution.

Embodiment 29 provides the method of any one of Embodiments 27-28,wherein the precursor for the biopolymer is about 2 mg/mL to about 8mg/mL of the pre-polymer solution.

Embodiment 30 provides the method of any one of Embodiments 27-29,wherein the pre-polymer solution further comprises a surfactant tomaintain the graphene in a non-agglomerated state during thepolymerization.

Embodiment 31 provides the method of Embodiment 30, wherein thesurfactant is about 5 mg/mL to about 15 mg/mL of the pre-polymersolution.

Embodiment 32 provides the method of any one of Embodiments 27-31,wherein the pre-polymer solution further comprises gelatin.

Embodiment 33 provides the method of Embodiment 32, wherein the gelatinis about 0.5 mg/mL to about 10 mg/mL of the pre-polymer solution.

Embodiment 34 provides the method of any one of Embodiments 27-33,wherein the pre-polymer solution further comprises PEG.

Embodiment 35 provides the method of any one of Embodiments 27-34,wherein the polymerizing comprises exposing the pre-polymer solution toa crosslinking solution.

Embodiment 36 provides the method of Embodiment 35, wherein thecrosslinking solution comprises an aqueous Ca²⁺ solution.

Embodiment 37 provides the method of any one of Embodiments 35-36,wherein the polymerization comprises injecting the pre-polymer solutioninto the crosslinking solution.

Embodiment 38 provides the method of any one of Embodiments 35-37,wherein the polymerization comprises exposing the pre-polymer solutionto the crosslinking solution in a microfluidic device.

Embodiment 39 provides a method of using the matrix-encapsulated cell ofany one of Embodiments 1-25, the method comprising:

-   -   detecting electrical signals from or sending electrical signals        to the one or more cells through the encapsulating polymer        matrix.

Embodiment 40 provides the method of Embodiment 39, wherein detectingthe electrical signals from the one or more cells comprises detectingresponses of the one or more cells to chemical or mechanical stimulusapplied to the one or more cells.

Embodiment 41 provides the matrix-encapsulated cell, fiber, or method ofany one or any combination of Embodiments 1-40 optionally configuredsuch that all elements or options recited are available to use or selectfrom.

What is claimed is:
 1. A method of making one or morematrix-encapsulated cells, the method comprising: polymerizing apre-polymer solution to form the one or more matrix-encapsulated cells,the pre-polymer solution comprising one or more cells, graphene, and aprecursor for a biopolymer; wherein the one or more matrix-encapsulatedcells comprise an encapsulating polymer matrix comprising thebiopolymer, and the graphene, wherein the graphene directly contacts atleast some of the one or more cells, and the encapsulating polymermatrix is electrically conductive.
 2. The method of claim 1, wherein thegraphene is about 10 mg/mL to about 30 mg/mL of the pre-polymersolution.
 3. The method of claim 1, wherein the precursor for thebiopolymer is about 2 mg/mL to about 8 mg/mL of the pre-polymersolution.
 4. The method of claim 1, wherein the pre-polymer solutionfurther comprises a surfactant.
 5. The method of claim 4, wherein thesurfactant is about 5 mg/mL to about 15 mg/mL of the pre-polymersolution.
 6. The method of claim 4, wherein the surfactant comprisespolyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitanmonopalmitate, polyoxyethylene (20) sorbitan monostearate,polyoxyethylene (20) sorbitan monooleate, polyethylene glycol (PEG),bovine serum albumen (BSA), or a combination thereof.
 7. The method ofclaim 4, wherein the surfactant maintains the graphene in anon-agglomerated state during the polymerization.
 8. The method of claim1, wherein the pre-polymer solution further comprises gelatin.
 9. Themethod of claim 8, wherein the gelatin is about 0.5 mg/mL to about 10mg/mL of the pre-polymer solution.
 10. The method of claim 1, whereinthe pre-polymer solution further comprises PEG.
 11. The method of claim1, wherein the polymerizing comprises exposing the pre-polymer solutionto a crosslinking solution.
 12. The method of claim 11, wherein thecrosslinking solution comprises an aqueous Ca²⁺ solution.
 13. The methodof claim 11, wherein the polymerization comprises injecting thepre-polymer solution into the crosslinking solution.
 14. The method ofclaim 11, wherein the polymerization comprises exposing the pre-polymersolution to the crosslinking solution in a microfluidic device.
 15. Themethod of claim 1, wherein the encapsulating polymer matrix is a fiber,a fibrous mat, a membrane, or a film.
 16. The method of claim 1, whereinthe encapsulating polymer matrix is a fiber that has a diameter of about1 micron to about 100 microns and a length that is greater than thediameter.
 17. The method of claim 1, wherein the biopolymer comprisesgelatin, chitosan, polycaprolactone, alginate, a polysaccharide, or acombination thereof.
 18. The method of claim 1, wherein the biopolymercomprises calcium alginate.
 19. The method of claim 1, wherein theencapsulating polymer matrix is less than or equal to 1 wt % grapheneoxide and wherein the encapsulating polymer matrix is less than or equalto 1 wt % reduced oxidized graphene.
 20. The method of claim 1, whereinthe one or more cells are one or more living cells.